We have shown previously that the ecf gene of E. coli is intimately associate with the active transport of amino acids and certain simple monosaccharides. Because of its importance in cellular processes, biochemical characterization of this energy coupling factor (ECF) will be pursued. The structural relationship of the ECF to beta-cystathionase will be investigated immunochemically. Reconstitution experiments are proposed to investigate the biochemical role of this protein in active transport. Experiments are proposed to tackle the problem of the energetics of the so-called shock-sensitive transport system. The direct energy donor will be identified both biochemically and genetically. The possible involvement of cytoplasmic factors in the energization of these transport systems will be investigated. Mutants pleiotropically defective in these transport systems will be sought for by the isotope suicide method. The role of binding proteins in active transport will be studied in the membrane vesicle systems. The main objective of this research proposal is to broaden our knowledge concerning the molecular organization of the energy transduction machinery and the mechanism of the coupling of metabolic energy to active transport.